The enormous increase in demand for synthetic oligonucleotides, fueled by the advances in DNA technology over the last decades, has been accelerated by recent progress in sequencing and decoding whole genomes, particularly the human genome. A number of methods in molecular biology and DNA based diagnostics to amplify, detect, analyze and quantify nucleic acids are dependent on chemically synthesized oligonucleotides. For instance, oligonucleotides are widely used as primers in PCR and in sequencing, and as probes in homogeneous assays or as components of microarrays. Synthetic oligonucleotides are also increasingly used in therapeutics as active pharmaceutical ingredients. For instance, oligonucleotides are used as antisense reagents with the aim to block the expression of certain proteins, as transcription factor inhibitors to block the translation of genetic information into messenger RNA, as aptamers to selectively inhibit proteins, as siRNA reagents to destroy the messenger RNA of a specific target, or as snRNA reagents to interfere in several aspects of the regulation of protein expression. Synthetic oligonucleotides are also used to store information in approaches towards molecular computing and in brand protection strategies.
A variety of methods have been developed for the chemical synthesis of oligonucleotides. The most prominent and in a commercial context almost exclusively used method is the automated solid phase synthesis using phosphoramidite chemistry, as disclosed by Caruthers et al. U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418, and Köster et al. U.S. Pat. No. Re. 34,069, and described by McBride et al. (1983) Tetrahedron Letters 24:245-248 and Sinha et al. (1983) Tetrahedron Letters 24:5843-5846. The nucleoside phosphoramidite mediated synthesis of oligonucleotides has been reviewed by Beaucage et al. (1992) Tetrahedron 48:2223-2311; (1993) Tetrahedron 49:6123-6194, and Tsukamoto et al. (2005) Frontiers in Organic Chemistry 1:3-40. In this method nucleotides are sequentially attached to a solid support in a predetermined order via their nucleoside phosphoramidite derivatives. Each attachment is conducted as a series of reaction steps, collectively called a synthesis cycle, which are performed in an automated synthesis machine, i.e. a synthesizer, which delivers the required reactants to the solid support in a pre-programmed manner. In the most common version of the phosphoramidite mediated synthesis of oligonucleotides phosphoramidites (1) are employed.

In (1) the 5′-hydroxyl function of the nucleoside is protected with a dimethoxytrityl (DMT)-protective group, which constitutes the front end protective group in the synthesis of oligonucleotides on a solid support. Basepg represents a nucleobase wherein the exocyclic amino group is protected with a base-labile protective group, e.g. Basepg represents N6-benzoyladenine, N4-acetylcytosine, N2-isobutyrylguanine or thymine, and R represents either a hydrogen atom, a protected hydroxyl group, an alkoxy group, e.g. R represents a methoxy group or a 2-methoxyethoxy group, or a fluorine atom. The synthesis cycle for the attachment of a nucleotide to the support via a phosphoramidite (1) includes the following reactions:                1. Front end deprotection/Detritylation                    Treatment of the solid support with a solution of a strong protic acid in an unpolar solvent, e.g. trichloroacetic acid in dichloromethane or dichloroacetic acid in toluene, to remove the DMT protective group in order to generate an unprotected hydroxyl group on the support.                        2. Phosphoramidite coupling                    Treatment of the support with a mixture of (1) and an activator in acetonitrile to covalently bind the nucleotide of the phosphoramidite to the support. 1H-Tetrazole, 4,5-dicyanoimidazole, 5-ethylthio-1H-tetrazole and 5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole are commonly employed activators. The resultant product is a nucleoside phosphite triester.                        3. Phosphite oxidation                    Treatment of the support with an oxidizing solution, preferably a solution of iodine, a mild base and water in a dipolar aprotic solvent, e.g. a 0.1 M iodine solution in pyridine/water/THF, to oxidize the phosphite triester to a phosphate triester. The application of a thioating reagent, e.g. 3H-1,2-benzodithiole-3-one 1,1-dioxide (‘Beaucage reagent’) or phenylacetyl disulfide in the phosphite oxidation steps results in the formation of respective thiophosphate triesters.                        4. Capping                    Treatment with a capping reagent which blocks unreacted functional groups on the support in order to prevent their participation in further coupling reactions. Most commonly a mixture of acetic anhydride with a catalyst in a dipolar aprotic solvent is employed, optionally supplemented by a mild base, e.g. a solution of acetic anhydride, N-methylimidazole and pyridine 1/1/1, v/v, in THF. Capping may also be performed before the oxidation step in the synthesis cycle. The capping reaction can be omitted in case of high coupling yields.                        
For small synthesis scales, i.e. scales of 1 μmol or less, the detritylation, oxidation and capping reactions can be performed in less than one minute. The time required for the coupling reaction depends on the type of phosphoramidite, on the activator employed, and the concentrations of phosphoramidite and activator. The coupling time may range from less than one minute for DNA phosphoramidites to several minutes for RNA phosphoramidites. E.g. appr. 5 minutes coupling time are required for RNA phosphoramidite couplings wherein the 2′-protective group is tert-butyldimethylsilyl and a 0.25 M solution of 5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole is applied as the activator.
The solid support of the synthesis typically consists of a derivatized inorganic or organic polymer, e.g. derivatized controlled pore glass (CPG) or polystyrene, with a linker to the first nucleoside, which is cleavable with base. After the completion of the desired number of synthesis cycles the linker is cleaved with base, typically an aqueous solutions of ammonia or methylamine, and the released oligonucleotide is separated from the support and further processed. Such processing includes the removal of the nucleobase and phosphate protective groups with base, which may be performed simultaneously with the cleavage of the linker to the support, the removal of protective groups on the 2′-hydroxy function of ribonucleosides, and the purification of the oligonucleotide by precipitation, desalting and/or chromatography.
The above method has been extensively applied in research laboratories as well as for the custom synthesis of oligonucleotides in commercial laboratories, or at larger scales, e.g. at scales of 1 mol or more, for the synthesis of therapeutic oligonucleotides. The method has, however, inherent weaknesses, which are based on the properties of the DMT protective group.
The cleavage of the DMT-group with acids is a reversible reaction and the primary cleavage product, i.e. the DMT-cation, can therefore reattach itself to the hydroxyl group on the support resulting in incomplete detritylations. Any non-deprotected hydroxyl groups cannot participate in the following phosphoramidite coupling reaction, but are most likely detritylated in the following synthesis cycle. As a result, a deletion sequence is built on the support, i.e. an oligonucleotide wherein one or more of the nucleotide units in the middle of the chain are omitted. Such a deletion sequence is cleaved from the solid support at the end of the synthesis and further processed together with the desired full-length product oligonucleotide. Most deletion sequences have very similar properties as the desired oligonucleotide products, because they differ from the product only in one missing nucleotide unit. They are therefore difficult to remove from the product through conventional purification techniques. Crude (unpurified) oligonucleotide synthesis products typically have deletion sequences as prominent impurities. Deletion sequences are particularly visible as ‘n−1’ signals in anion-exchange chromatograms. The side product representing the ‘n−1’ signal of an oligonucleotide synthesis has been analyzed by Fearon et al. (1995) Nucleic Acids Res. 23:2754-2761, and by Temsamani et al. (1995) Nucleic Acids Res. 23:1841-1844, and has been shown to consist of mixtures of all possible deletion sequences of the respective full-length product sequences.
The main strategy applied today to drive the deprotection of the DMT-group to completion is the removal of the DMT-cation from the reaction equilibrium. On solid supports this is accomplished through multiple replacements of the reaction solution with fresh reagent solution, often through permanent washing of the support with fresh detritylating reagent during the reaction. Detritylation efficiencies in excess of 99% are often achieved through this technique, but relatively large volumes of the aggressive and harmful reagent solutions are consumed. Also, although the detritylation is nearly complete, a small fraction of the DMT-groups always remains undetritylated in every synthesis cycle, giving rise to an accumulating pool of deletion sequences, which then become impurities in the crude oligonucleotide synthesis product.
Another drawback of the method is the repeated exposition of the growing oligonucleotide chain to strong protic acid. Protic acids cause side reactions, in particular depurinations, which result in chain scission during the deprotection with base at the end of the synthesis. Although the current detritylating reagents are optimized with respect to high detritylation efficiency versus low depurination rates the side reaction still takes place in each cycle. In particular, purines, which are introduced in the early synthesis cycles, have multiple exposures to the acidic reagent and are partially depurinated at the end of the synthesis.
Another drawback of the use of the DMT group for front-end protection is the inherent susceptibility of this group to partial cleavage during the phosphoramidite coupling reaction. The coupling reaction is performed in the presence of an excess of slightly acidic activators, which partially cleave the DMT-group of the coupling product on the support. The resulting cleavage products can participate in further coupling reactions which in turn leads to the formation of sequences with inserted additional nucleosides, i.e. longer oligonucleotides.
All of the above drawbacks become more severe with increasing chain length of the synthesized oligonucleotide. Deletions sequences and sequences with additional inserted nucleosides accumulate to a larger extend in longer sequences than in shorter sequences and are also more difficult to remove from longer sequences than from shorter sequences. The consumption of harmful and environmentally undesired solvents and acidic reagents is linearly related to the number of synthesis cycles in the preparation of an oligonucleotide. The exposure time of a given nucleotide unit of the growing oligonucleotide to acid also relates linearly to the number of synthesis cycles. The total exposure of the growing chain to acid, as determined by adding the number of exposed nucleotide units in the growing chain at each synthesis cycle to an exposure sum, however, increases in a much stronger than linear manner with increased cycle number and may pose a limit to the length of oligonucleotides, which can be synthesized in practical terms with the method described above.
Several attempts were described to utilize alternative front-end protective groups instead of the DMT-group in a phosphoramidite mediated solid phase synthesis. In one approach a silyl group, in particular the bis(trimethylsilyloxy)cycloundecanoxysilyl group, is applied, as described by Scaringe et al. (1998) J. Am. Chem. Soc. 120:11820-11821 for phosphoramidites with methyl phosphate protective groups in the synthesis of RNA oligonucleotides. The silyl groups are removed with the highly toxic hydrofluoride-triethylamine complex or similar hydrofluoride reagents and are not compatible with the common β-cyanoethyl phosphate protective group. In another approach the monomethoxytritylthio group is applied instead of the DMT-group, as described by Seio et al. (2001) Tetrahedron Letters 42:8657-8660 in the synthesis of the 5-mer dT5. The monomethoxytritylthio group is removed under oxidative conditions with a solution of 0.1 M iodine in acetonitrile/pyridine/water (10/9/1, v/v) and allows simultaneous deprotection and oxidation within a synthesis cycle. A demonstration of the usefulness of this approach was limited to the synthesis of a single, short sequence dT5, which was obtained in a relatively low yield. In another approach the 2-(levulinyloxymethyl)-5-nitrobenzoyl group is applied instead of the DMT-group, as described by Kamaike et al. (1997) Tetrahedron Letters 38:6857-6860 in the synthesis of an RNA 8-mer. The 2-(levulinyloxymethyl)-5-nitrobenzoyl group is removed on the support in a two-step procedure consisting of a treatment with a solution of hydrazine hydrate for 15 minutes followed by a treatment with a solution of imidazole for 5 minutes. The rather long deprotection time, the need for two different deprotection reagents and the carcinogenic nature of the first reagent are drawbacks of this method.
In some known methods alternative front end protective groups to the DMT-group are applied in the phosphoramidite method, which are cleaved under basic conditions. In one example the 2-dansylethoxycarbonyl group was applied instead of the DMT-group for deoxyribonucleoside phosphoramidites with p-nitrophenylethyl phosphate protective groups as described by Bergmann et al. (1994) Helv. Chim. Acta 77:203-215 for the synthesis of homopolymers of dA, dC, dG and dT with a length up to 10 nucleotide units. The 2-dansylethoxycarbonyl group is cleaved with a 0.1 M solution of DBU in acetonitrile within 140 sec., presumably through a β-elimination mechanism. In another example the (2-cyano-1-phenyl)ethoxycarbonyl group was applied instead of the DMT-group for ribonucleoside phosphoramidites with p-nitrophenylethyl phosphate protective groups as described by Münch et al. (1997) Nucleosides & Nucleotides 16:801-808. The (2-cyano-1-phenyl) ethoxycarbonyl group is cleaved with a 0.1 M solution of DBU in acetonitrile within 20 sec., presumably through a β-elimination mechanism. The highly reactive 1-phenylacrylonitrile is formed as a by-product in this approach, which may react with nucleobases. In another approach the p-phenylazophenyloxycarbonyl group is applied instead of the DMT-group for deoxyribonucleoside phosphoramidites with methyl phosphate protective groups as described by Seliger et al. (1985) Nucleosides & Nucleotides 4:153-155 for the synthesis of short poly-dt sequences. The p-phenylazophenyloxycarbonyl group is removed through a two-step reaction sequence, which involves treatment with a mixture of 2-cyanoethanol, triethylamine and water 1/1/1, v/v, for 1 minute, followed by a treatment with a mixture of DBU and pyridine 1/1, v/v, for 1 minute. The proposed mechanism of cleavage involves a transesterification to generate the respective β-cyanoethoxycarbonyl compounds followed by β-elimination. A rather low yield of poly-dT sequences with lengths up to 9-nucleotides was obtained. In another approach substituted aryloxycarbonyl groups, preferable 3-trifluoromethylphenyloxycarbonyl groups, were applied instead of the DMT-group for deoxyribonucleoside phosphoramidites with β-cyanoethyl phosphate protective groups as described by Sierzchala et al. (2003) J. Am. Chem. Soc. 125:13427-13441; U.S. Pat. No. 6,222,030. The 5′-aryloxycarbonyl groups are removed with a buffered solution of lithium hydroxide, hydrogen peroxide and m-chloroperbenzoic acid in a solvent cocktail containing 2-amino-2-methyl-1-propanol and water at pH 9-10 in this method. The cleavage of the 5′-protective groups is presumably accomplished with peroxyanions formed in the deprotection solution, which act as strong nucleophiles. In this approach the phosphite oxidation reaction of the synthesis cycle is carried out at the same time as the front end deprotection reaction, because the peroxy anions also represent mild oxidizing agents. The method has also been applied in the simultaneous synthesis of a multitude of oligonucleotides on flat surfaces, i.e. in the synthesis of microarrays. The deprotection solution containing peroxyanions is, however, not stable and needs to be freshly prepared every day. In another approach the DMT group was replaced by the 9-fluorenylmethyloxycarbonyl (Fmoc) group for ribonucleoside phosphoramidites with β-cyanoethyl phosphate protective groups as described by Lehmann et al. (1989) Nucleic Acids Res. 17:2379-2390. The Fmoc group is cleaved with a 0.1 M solution of DBU in acetonitrile in this approach, presumably via a β-elimination mechanism. In this approach the highly reactive dibenzofulvene is generated as a by-product, which may lead to reactions with nucleobases or may polymerize and cause clogging of solvent lines or filters on a synthesizer. Also, the stepwise coupling yields were limited to 96% in the described synthesis, which is not high enough for the synthesis of long oligonucleotides.
In all of the methods cited above wherein alternative front end protective groups to the DMT-group are cleaved under basic conditions the respective protective groups represent alkyloxycarbonyl- or aryloxycarbonyl groups. Also, in all of these methods the mechanism of cleavage for the respective protective group is either cleavage via β-elimination or cleavage with strong nucleophiles. In the present invention other alternative protective groups to the DMT-group are disclosed, which represent acyl groups, and which are cleaved with organic bases like primary amines or phenolates. Furthermore, the present invention discloses phosphoramidites for the solid phase synthesis of oligonucleotides wherein the front end protective group is a base labile protective group, which is an acyl group, and which is cleaved with an organic base like a primary or secondary amine. Also, the present invention discloses methods for the synthesis of oligonucleotides, which are based on the application of the above phosphoramidites in the phosphoramidite-mediated synthesis of oligonucleotides on solid supports.